The importance of accurately measuring cholesterol levels in blood is well known. The federal government, in combination with more than twenty health organizations, has launched an aggressive campaign, through the National Cholesterol Education Program, to convince physicians and the general population of the dangers of high cholesterol levels in the blood. All persons are urged to have their cholesterol levels checked, and specific treatments are recommended based on the precise measured cholesterol level. In addition, treatments are not based solely on the total cholesterol level, but instead, on the level of LDL cholesterol. LDL cholesterol appears to be the major cause of clogged arteries, whereas HDL cholesterol aids in removing cholesterol deposits. A separate, and more expensive test is required to determine the level of LDL cholesterol and it is usually not conducted unless the measured total cholesterol level is at the borderline or high risk levels.
The most common methods for measuring cholesterol levels are notoriously inaccurate and the standard practice is to repeat the measurement a number of times when high levels are detected on the first measurement. Inaccuracies of 5% or more have been found in nearly half of the measurements made by testing laboratories and 15% of the measurements were inaccurate by an amount greater than 10%. These inaccuracies are inherent in the current measurement methods which require considerable handling of the blood and certain presumptions about the ratios of its constituent parts.
Direct quantization of lipoprotein cholesterol is usually achieved by enzymatic assay of the individual lipoproteins, which are separated by ultracentrifugation, electrophoresis, or selective precipitation. There is great variability among the available separation methods in terms of accuracy, convenience, and cost. Generally, the most accurate methods are those involving ultracentrifugation, but these are very time consuming and expensive and therefore not suitable for largescale population studies. The most widely used alternative is an indirect method introduced by W. T. Friedewald, R. I. Levy, and D. S. Fredrickson, Estimation of the Concentration of Low-Density Lipoprotein Cholesterol in Plasma, Without Use of the Preparative Ultracentrifuge, Clin. Chem. 18, 499-502 (1972). In this procedure, plasma triglyceride (TG) and total cholesterol (TC) are measured by enzymatic assay. To a separate aliquot of plasma is added one of several reagents which selectively precipitates VLDL and LDL. After removing the precipitate by centrifugation, the supernatant is assayed for cholesterol to provide a measure of HDL cholesterol (HDL-C). An estimate of VLDL cholesterol (VLDL-C) is then made by dividing the plasma triglyceride level by five. The LDL cholesterol (LDL-C) concentration is then calculated by difference: LDL-C=TC-(HDL-C+VLDL-C). Although this method is relatively rapid and inexpensive, there are several steps where experimental error can be introduced, particularly in the precipitation step. In addition, the accuracy of the analysis depends on the assumption that VLDL-C can be reliably estimated as one fifth the concentration of plasma triglyceride.
When fasting samples are used, this is generally true, but other formulas have also been suggested to give more accurate values as described by D. M. DeLong, E. R. DeLong, P. D. Wood, K. Lippel, and B. M. Rifkind, A Comparison of Methods for the Estimation of Plasma Low- and Very Low-Density Lipoprotein Cholesterol, J. Am. Med. Assoc. 256, 2372-2377 (1986).
It has also been shown that the major lipoprotein constituents could be further subdivided into subclasses based on further refinement of particle densities. Krauss et al, J. Lipid Research 23, 97-104 (1982), Atger et al., Clinical Chemistry 37, 1149-1152 (1991). The distribution of these subclasses within a major lipoprotein group may in itself provide further insight into risk analysis of CHD. Stossel et al, JAMA 260, 1917-1921 (1988). However, previous methods of determining the distribution profile of subclasses have been time consuming and unable to determine a number of subclass concentrations simultaneously.